ANKIB1 is an RBR domain-containing protein. It has been classified as an E3 ubiquitin ligase for a long time. However, little is known about its E3 ligase activity and related ubiquitin-conjugating enzymes (E2s). We investigated the expression pattern, cellular localization, and brain distribution of ANKIB1. Immunoblotting showed that human cancer cell lines had different expression patterns of ANKIB1 with virus-transformed immortal lymphocytes and human brain. Immunocytofluorescence analysis indicated that ANKIB1 was mainly localized in the cytoplasm, and immunohistochemistry staining demonstrated that ANKIB1 was highly expressed in the rat brain cortex but seldom expressed in the basal ganglia. We then performed coimmunoprecipitation, in vitro and in vivo ubiquitination assay to determine whether ANKIB1 functions as an E3 ligase. A panel of class I and class III E2s were tested, of which UbcH8, UbcH10, and UbcH13 were confirmed to interact with ANKIB1. Moreover, ANKIB1 showed E3 ligase activity both in vivo and in vitro, but it promoted protein ubiquitination more efficiently in vivo. These data suggest that ANKIB1 acts as a partner of an E3 ligase complex, and may play important roles in regulating tumorigenesis and brain cortex function.